Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

Articles Written By Tim Bushnell, PhD

Combining Flow Cytometry With Plant Science, Microorganisms, And The Environment

By: Tim Bushnell, PhD

My first introduction to flow cytometry was talking to a professor who’d brought one on a research cruise to study phytoplankton. It was only later that I was introduced to the marvelous world that’s been my career for over 20 years.   In that time, I’ve had the opportunity to work with researchers in many different areas, exposing me to a wide variety of cell types and more important assays. What continues to amaze me is the number of different parameters we can measure, not just the number of fluorochromes, but the information we can extract from samples – animal, vegetable…

Common Numbers-Based Questions I Get As A Flow Cytometry Core Manager And How To Answer Them

By: Tim Bushnell, PhD

Numbers are all around us.  My personal favorite is ≅1.618 aka ɸ aka ‘the golden ratio’.  It’s found throughout history, where it has influenced architects and artists. We see it in nature, in plants, and it is used in movies to frame shots. It can be approximated by the Fibonacci sequence (another math favorite of mine). However, I have not worked out how to apply this to flow cytometry.  That doesn’t mean numbers aren’t important in flow cytometry. They are central to everything we do, and in this blog, I’m going to flit around numbers-based questions that I have received…

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

4 Critical Rules For Spectral Unmixing

By: Tim Bushnell, PhD

Spectral unmixing is the mathematical process by which a spectrum is broken down into the abundances of the different fluorochromes that make up the observed spectrum. This was described in the paper by Novo et al., (2013), which presented a generalized model for spectral unmixing of flow cytometry data. Of course, like compensation in traditional fluorescent flow cytometry, there are important rules to observe regarding the controls that are used to unmix the sample. If you need a refresher on the rules for TFF compensation, you can read about them here.    This blog will discuss the generalized process of spectral unmixing…

How To Buy A Flow Cytometer - What You Need To Evaluate From A To Z

By: Tim Bushnell, PhD

So you have the money to buy a flow cytometer. Is it a sorter? Or perhaps a spectral analyzer? No wait, maybe an imaging mass cytometer?  Big or small?  What to choose?  How to choose?  More importantly, once you sign the contract to purchase the instrument, you don’t want to be struck with buyers remorse.  It is indeed a big decision and we have the best advice for you to consider before making the purchase. Let’s discuss some of the steps you should take to prevent buyers remorse and ensure you are getting the best instrument for your needs.  Do…

How small can you go? Flow cytometry of bacteria and viruses

By: Tim Bushnell, PhD

Flow cytometers are traditionally designed for measuring particles, like beads and cells. These tend to fall in the small micron size range. Looking at the relative size of different targets of biological interest, it is clear the most common targets for flow cytometry (cells) are comparatively large (figure 1). Figure 1:  Relative size of different biological targets of interest. Image modified from Bioninja.    In the visible spectrum, where most of the excitation light sources reside, it is clear the cells are larger than the light. This is important as one of the characteristics that we typically measure is the amount…

What Is Spectral Unmixing And Why It's Important In Flow Cytometry

By: Tim Bushnell, PhD

As the labeled cell passes through the interrogation point, it is illuminated by the excitation lasers. The fluorochromes, fluoresce; emitting photons of a higher wavelength than the excitation source. This is typically modeled using spectral viewers such as in the figure below, which shows the excitation (dashed lines) and emission (filled curves) for Brilliant Violet 421TM (purple) and Alexa Fluor 488Ⓡ (green).  Figure 1: Excitation and emission profiles of BV421TM and AF488Ⓡ  In traditional fluorescent flow cytometry (TFF), the instrument measures each fluorochrome off an individual detector. Since the detectors we use — photomultiplier tubes (PMT) and avalanche photodiodes (APD)…

How To Extract Cells From Tissues Using Laser Capture Microscopy

By: Tim Bushnell, PhD

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…